There are different practices in the operational procedures for use in the removal, recovery, deprotection and/or purification of biopolymers from a solid support. For example, when the biopolymer is newly synthesized DNA, the removal of DNA from the solid support, after completion of its synthesis, is accomplished by cleaving the chemical-bond that anchors the DNA polymer to the solid polymer matrix. In most applications, the chemical bond is an ester bond between a carboxylic acid and an alcohol (the 3'-hydroxyl group on the deoxyribose moiety of the sugar), which can be cleaved by hydrolysis using, for example, concentrated ammonia. The cleavage reaction is generally 80 to 100% complete within 1 hour at room temperature.
When the cleavage reaction (for the removal of the DNA) is done on DNA synthesizers that are equipped with an automatic cleavage feature (e.g., such as models presently available from Applied Biosystems or Milligen-Biosearch), the reaction is accomplished by pulsating concentrated ammonia through the synthesis column for about 1 hour, using an inert gas as propellant. The reaction time for the removal can be programmed into the machines, but is usually between 1 and 2 hours with a total aqueous ammonia consumption of 0.5 to 1.5 mL. The ammonia solution containing the recovered DNA polymer is collected in a vial attached to the machine. (During the cleavage operation the machines cannot be used for any other synthesis operations.) The recovered DNA polymer is next deprotected, purified and isolated, either in other instruments or by manual techniques.
Most DNA synthesizers are not equipped with the automatic cleavage feature for the DNA synthesis columns, and the cleavage reaction is instead done manually by laboratory personnel. DNA synthesizers without this feature are the most common, since they cost substantially less. The introduction of ammonia into the DNA synthesis columns can be done by many different manual procedures. The solid support in the column can be transferred to a vial containing aqueous ammonia, the vial sealed and allowed to stand at room temperature for about 1 hour; the vial can be heated to speed up the cleavage. The DNA is recovered in the supernatant, which is then separated from the solid support. The supernatant then undergoes further deprotection and purification procedures. This procedure for removing DNA from the support was commonly used several years ago when DNA laboratories packed their own DNA synthesis columns. Today most laboratories buy columns from chemical supply houses.
Nowadays, a modification of the above procedure is often used. Typically the column is removed from the synthesizer and a syringe is filled with aqueous ammonia (1 to 3 mL) and attached to the column. An empty syringe is fitted to the other end of the column to receive the ammonia that has passed through the column. From this point, the procedure may follow one of two methods. One method is to flush the entire contents of the feeding syringe through the column into the receiving syringe over the course of 10 to 30 seconds, and then allow the syringe unit to stand for 5 to 10 minutes. This is followed by a flush through the column in the other direction (the receiving syringe is now being used as feeding syringe, and the first feeding syringe now acts as the receiving syringe). This flushing back and forth should be done for at least 1 hour to achieve a nearly complete DNA removal from the DNA synthesis column.
A second method is to mimic the cleavage procedures used by the DNA machines. The contents of the feeding syringe (1 to 3 mL) are slowly pulsated through the column and collected in the receiving syringe. Since it is not possible to feed a very small amount of ammonia through the column continuously for 1 hour at a volume of 1 to 3 mL, a small amount (0.1 to 0.2 mL) is fed into the column, and the column is allowed to stand for 5 to 10 minutes before another addition of the same amount of ammonia. This procedure is repeated several times during the course of about 1 hour. Thus, about 0.1 to 0.2 mL of ammonia should be added to the column every 5 to 10 minutes for about 1 hour to achieve a nearly complete removal of the DNA attached to the DNA synthesis column. The DNA is recovered in the aqueous ammonia, collected in the receiving syringe. The DNA is ready for further deprotection and purification.
The DNA that has been removed and recovered from the DNA synthesis column contains a lipophilic protecting group at the 5'-end of the sequence (dimethoxytrityl group, also called the "DMT-group"), unless that group has been removed on the DNA synthesizer. Removal of the DMT-group is an option on the DNA synthesizer. Depending on the research and purification needs, the researcher may, or may not, choose this option. After the DNA, with or without the DMT-group, has been removed from the DNA synthesis column, it is deprotected at the bases and at the phosphorus using heated ammonia. The DMT-group is stable in ammonia and is not removed in the deprotection protocol when the DNA containing that group (i.e., "DMT-DNA") is heated at 55.degree. C. for 5 to 24 hours to remove remaining protecting groups on the DNA polymer.
The DMT-DNA can be purified using affinity chromatography or reversed phase chromatography. High performance liquid chromatography ("HPLC") is often used for the purification, eluting the DMT-DNA on a reversed phase HPLC column using acetonitrile/triethylammonium acetate as eluent. The DMT-group is then removed under acidic conditions (80% aqueous acetic acid at room temperature for 15 to 60 minutes) and the fully deprotected DNA can be used after concentration of the eluent.
Recently, an alternative to the expensive HPLC isolation of DNA was introduced; the procedure is called cartridge purification. It is based on the same principle as reversed phase HPLC, in that the purification cartridge contains a lipophilic solid matrix to retain the DMT-DNA in the cartridge during the purification, while organic material from the deprotection procedure and non-DMT-DNA is washed off the cartridge. The cartridge purification does not have the resolution power of HPLC purification, but is sufficient for most DNA applications. There is one major difference between using HPLC purification versus cartridge purification; instead of removing the DMT-DNA from the cartridge, as is usually done on HPLC purification, the DMT-group is removed from the DMT-DNA with acid while the DMT-DNA is still on the cartridge. Then the fully deprotected DNA is then eluted off the cartridge with acetonitrile (20%) in water. The DNA is ready to be used after the solvent is evaporated.
The DNA cartridge purification is typically done by feeding reagents and solvents into the cartridge, shaped as a column, in a prescribed order, using a syringe. The cartridge is first washed with acetonitrile, then with triethylammonium acetate (1 to 2 M). The deprotected DMT-DNA is slowly applied to the cartridge in concentrated or diluted ammonia; it is generally recommended that the solution be passed through the cartridge at least 2 times. The cartridge is then washed with diluted ammonia to remove salts and non-DMT-DNA. The cartridge is next washed with water to remove the ammonia. The DMT-group is then removed with 1 to 2% trifluoroacetic acid by feeding the solution through the cartridge. The cartridge is then washed with water and the fully deprotected DNA is removed from the cartridge by aqueous acetonitrile. The DNA sample is concentrated and the DNA can be used.
This multi-step procedure is typically performed manually for each DNA sample. Because of the manipulations, this procedure involves a substantial amount of work and time. The solvents and reagents are fed by a syringe through the cartridge. The syringe has to be reloaded for each step. After several purifications, it can become a physical burden for the researcher to continue using the same thumb to push the syringe plunger in order to deliver the chemicals, or solvents, into the tightly packed cartridge. The cartridge purification takes approximately 15 to 20 minutes for each sample. In a laboratory that processes many samples, the time and the labor involved can be quite substantial.